The microsporidia are "emerging" human and veterinary pathogens that cause disease in both immunocompromized and immunocompetent hosts. Our efforts are directed at understanding the invasion mechanism of these organisms involving the polar tube and its associated structures which form an organelle critical for cell invasion. The polar tube serves as a vehicle for transmission of infection by piercing an adjacent host cell inoculating the sporoplasm directly into that cell. The biochemical components of this structure and the mechanism of its formation during invasion remain to be definitively determined. Study of the composition, formation and function of this organelle during germination and invasion should provide a basis for the development of new strategies for control of these important parasitic protists. We plan to use a proteomics (mass spectrometry) approach to the characterization of the proteins of the polar tube (PTPs) and spore wall (SWPs) of the microsporidia. Encephalitozoon cuniculi, a human pathogenic microsporidia, will be used for these studies as the availability of its complete genome facilitates this proteomic approach. Recombination cloning will be used to express and characterize the identified proteins as this will allow high throughput expression analysis. The molecular interaction(s) of PTP1 with itself and with other PTPs will be evaluated using yeast two hybrid and GST pull- down techniques. Our characterization of these structures at the molecular level will be complemented by ultrastructural studies investigating the development and location of PTPs and SWPs during morphogenesis and eversion of the polar tube. Cryoelectron microscopy and EM tomography will provide insights into the 3D structure of the polar tube and spore wall during eversion, providing answers to fundamental questions on the organism of these structures that have not been resolved by traditional EM. For example, our observations suggest that the polar tube may be extracellular in the spore. We believe that the information gained by delineating, in detail, the function and components of the polar tube will eventually lead to novel therapeutic interventions that could limit or interdict the transmission of these emerging pathogens. RELEVANCE. These are new pathogens for which there are limited effective drugs. Studies of how these organisms cause infection should result in the development of new therapeutic agents.